Proteomic Profiling and T Cell Receptor Usage of Abacavir Susceptible Subjects

Abstract

Type B adverse drug reactions (ADRs) represent a significant threat as their occurrence arises unpredictable and despite proper application of the drug. The severe immune reaction Abacavir Hypersensitivity Syndrome (AHS) that arises in HIV⁺ patients treated with the antiretroviral drug Abacavir (ABC) strongly correlates to the presence of the human leukocyte antigen (HLA) genotype HLA-B*57:01 and discriminates HLA-B*57:01⁺ HIV⁺ patients from ABC treatment. However, not all HLA-B*57:01⁺ HIV⁺ patients are affected by AHS, implying the involvement of further patient-specific factors in the development of AHS. The establishment of a reliable assay to classify HLA-B*57:01 carriers as ABC sensitive or ABC tolerant allowed to investigate the T cell receptor (TCR) Vβ chain repertoire of effector cells and revealed Vβ6 and Vβ24 as potential public TCRs in ABC sensitive HLA-B*57:01 carriers. Furthermore, distinct effects of ABC on the cellular proteome of ABC sensitive and tolerant volunteers were observed and suggest enhanced activation and maturation of dentritic cells (DC) in ABC sensitive volunteers. Analysis of ABC-naïve cellular proteomes identified the T cell immune regulator 1 (TCIRG1) as a potential prognostic biomarker for ABC susceptibility and the involvement of significantly upregulated proteins, particularly in peptide processing, antigen presentation, interferon (IFN), and cytokine regulation.

Keywords: HLA-B*57:01; T cell receptor; abacavir; adverse drug reaction; hypersensitivity; proteome.

Figure 1

ABC concentrations in cell lysates following various incubation periods. LCL721.221 cells were cultured in the absence or presence or absence (ø ABC) of 50 µg/mL ABC in three technically independent replicates (n = 3) and ABC amounts were measured by UPLC-MS/MS.

Figure 2

Classification of HLA-B*57:01⁺ healthy volunteers as ABC sensitive (red) or ABC tolerant (blue). Depicted is the cytotoxic potential (% of dead cells) of CD8⁺ cells following ABC treatment of target cells. CTA was conducted in two biologically and technically independent replicates (n = 4). Dash line shows the threshold for classification.

Figure 3

Difference in relative TCR Vβ5, Vβ6, and Vβ24 mRNA expression in ABC-specific CD8⁺ cells compared to non-specific CD8⁺ cells of ABC sensitive and ABC tolerant HLA-B*57:01⁺ healthy volunteers. TCR Vβ repertoires of CD8⁺ cells of classified ABC sensitive and ABC tolerant healthy HLA-B*57:01 carriers were analyzed before and after 48 h of ABC-stimulation or unspecific stimulation. The relative Vβ mRNA expression was normalized to the constant region of the TCR and the difference in transcription in ABC-specific to non-specific CD8⁺ cells after 48 h was normalized to Vβ mRNA expression of naïve CD8⁺ cells. Bars and error bars represent mean ± SEM. Experiments were performed in four biologically and three technically independent replicates (n = 12). * p ≤ 0.05, ** p ≤ 0.01.

Figure 4

Principal component analysis (PCA) of proteins that were significantly altered (p < 0.05) in PBMCs of each three classified ABC sensitive (red) and ABC tolerant (blue) HLA-B*57:01⁺ healthy donors following ABC treatment. PBMCs were incubated without (ø) or with (+) ABC for 48 h in three biologically and three technically independent replicates (n = 9).

Figure 5

Differences in protein abundance in PBMCs of each three classified ABC sensitive and ABC tolerant HLA-B*57:01⁺ healthy donors following ABC treatment. The volcano plot illustrates significantly differentially abundant proteins after ABC treatment of three biologically and three technically independent replicates (n = 9). The log₂ fold change of ABC sensitive compared to ABC tolerant HLA-B*57:01⁺ volunteers is plotted against the −log₁₀ p-value. Proteins were regarded as regulated in ABC sensitive donors from factor ± 1.0 and p < 0.05. Downregulated proteins are labelled in green, unregulated proteins are colored in grey, and upregulated proteins are given in red.

Figure 6

Differences in protein abundance in untreated PBMCs of each three classified ABC sensitive and ABC tolerant HLA-B*57:01⁺ healthy donors. The volcano plot illustrates significantly differentially abundant proteins of three biologically and three technically independent replicates (n = 9). The log₂ fold change of ABC sensitive compared to ABC tolerant HLA-B*57:01⁺ donors is plotted against the −log₁₀ p-value. Proteins were regarded as regulated in ABC sensitive volunteers from factor ±1.0 and p < 0.05. Downregulated proteins are labelled in green, unregulated proteins are colored in grey, and upregulated proteins are given in red.

Figure 7

Protein-protein interaction network of significantly up- and downregulated proteins in ABC sensitive compared to ABC tolerant HLA-B*57:01 carriers and cellular processes they are involved in (based on GO/KEGG). The network was constructed by the STRING Database (Version 11.5) and visualized using Cytoscape (Version 3.8.2). Significant regulated proteins determined by permutation-based FDR and at least altered by factor log₂ + 1 were considered. Upregulated proteins are illustrated in red. Color intensity reflects the log₂ fold difference between ABC sensitive compared to ABC tolerant HLA-B*57:01 carriers.